Nuclear transfer nuclei from histone hypomethylated donor cells

ABSTRACT

The present invention provides a method of producing an animal embryo, the method comprising transferring from a nuclear donor cell which has been selected on the basis that it is histone hypomethylated at least a portion of the nuclear contents including at least the minimum chromosomal material able to support development into a suitable recipient cell.

The present invention relates to cloning procedures in which cell nucleiare transplanted into recipient cells. The nuclei are reprogrammed todirect the development of cloned embyros, which can then be transferredinto recipient females to produce foetuses and offspring or used toproduce embryonic cell lines.

All publications, patents and patent applications cited herein areincorporated in full by reference.

BACKGROUND

A fundamental question in cell and developmental biology concerns hownuclei progressively acquire differentiated functions. Although thenucleus of a fertilised egg is totipotent in that all of thedifferentiated cell types found in the adult organism can be derivedfrom it, this is not the case for the vast majority of somatic nuclei inthe adult animal. This limitation of the genomic potential of nuclei isprogressively acquired during embryonic and post-embryonic development.Although in most cells the DNA sequence content of nuclei remainsunchanged as development proceeds, the repertoire of genes that areexpressed in a given cell type becomes limited. It also becomes moredifficult to reactivate genes that are silenced in that cell type. Thislimitation is now known to reflect the imposition of epigeneticregulatory mechanisms on genes, especially through the assembly ofstable repressive nucleoprotein complexes in the differentiated cellnucleus. The molecular mechanisms necessary to stably repress genes aregradually established as embryogenesis and post-embryonic developmentproceed. Remarkably, the egg and oocyte can reverse this process ofrepression, disassembling repressive features of nuclear organisationand, in particular circumstances, recreating a state, of pluripotencyand even totipotency.

Covalent modifications to histone proteins have been proposed as thebasis for an epigenetic code capable of extending the informationpotential of primary DNA sequences [1]. This code could ‘mark’ thetranscriptional status of genes and also provide a plausibleself-templating mechanism to propagate chromatin status through DNAreplication and mitosis. Transcriptionally active euchromatin andinactive heterochromatin have been characterized by generalizeddifferences in histone modifications [2]. For example, there is a globalunder-acetylation (particularly of H4) in heterochromatin domains suchas those exemplified by the inactive X chromosome in mammals [3]. Inaddition, more subtle site-specific changes are also consistent featuresof euchromatin versus heterochromatin. For example, acetylation atlysine 12 in H4 appears to be a hallmark of heterochromatin [4, 5]whereas acetylation of lysine 9 in H3 represents a euchromatic imprintin Tetrahymena and many other organisms (reviewed in [6]). Methylationof H3 at lysine residues 4 or lysine 9 is reciprocally associated witheuchromatic or heterochromatic regions, respectively [7,1].

A discussion of methylation at lysine 9 of H3 in animals may be found inCowell et al. (2002) Chromosomsa, 111:22-36. Contrary to the findingspresented herein, Cowell et al. states that methylation at lysine 9 ofH3 represents one of the most robust histone modifications and suggeststhat it is almost permanent in nature.

Although an increasing number of factors involved in transmitting geneexpression patterns have been identified, we do not as yet know how, ata mechanistic level, transcriptional competence is conveyed to daughtercells. Polycomb (PcG) and Trithorax (TrxG) group proteins appear to becrucial for the clonal inheritance of the inactive and active state oftarget genes in diverse organisms [8, 9]. In addition, genes previouslycharacterized as modifiers of position effect variegation (PEV) can alsoinfluence the transmission of epigenetic information [10-14]. Theseinclude some structural components of heterochromatin, such as HP1(allelic to Su(var)2-5), as well as enzymes that modify histones, suchas the Suv39h HMTases [14-17].

Interest in the basic molecular mechanisms involved in the imposition ofepigenetic regulatory mechanisms on genes has been stimulated by theeconomic and medical implications of the cloning of animals by nucleartransfer from donor embryos and from adult cell nuclei. Unfortunately,the economic and medical exploitation of cloning technology has beenhampered by the extremely low efficiency of cloning from adult cellnuclei with most clones dying during gestation.

Somatic nuclei can be reprogrammed by nuclear transfer into enucleatedoocytes as originally described by Wakayama and colleagues in 1998.Although approximately 20-40% of renucleated oocytes develop to theblastocyst stage, in most case less than one percent result in live bornanimals suggesting that complete reprogramming is a rare event (reviewedin Yanagimachi, R. Mol Cell Endocrin. (2002) 187 p 241-248).Reprogramming can also be achieved by clear transfer into fertilisedmouse eggs (Modlinski, J. A. 1978. Nature 273 p. 466-467). Although thelatter technique results in tetraploidy of the resultant embryos, thetechnique itself is much simpler and more robust than traditionalcloning (our observations) and allows the assessment of the differencesin reprogram potential of multiple cell types.

Attempts to increase efficiency have included varying the source ofdonor nuclei. For instance, EP 930 009 describes the use of restingcells as nuclear donor cells whilst WO 99/53751 and Hoechedlinger andJaenisch (2002) Nature, 415: 1035 to 1038 describes the use oflymphocytes as nuclear donors. However, Hoechedlinger and Jaenisch(2002) found that the use of lymphocytes as nuclear donor cells wasrelatively inefficient and concluded that the efficiency was about tentimes lower than that from other donor cell populations. It wassuggested that the low efficiency could be due to inefficientreprogramming of the lymphocyte genome or differences in the sensitivityof the lymphocyte nuclei to the nuclear transfer protocol.

In view of the foregoing, it will be appreciated that there is a needfor an improved understanding of the mechanisms underlying epigeneticregulation and a need for new approaches towards improving theefficiency and success of nuclear transfer procedures.

THE INVENTION

The present invention is based on the discovery that cells which havehistone hypomethylation may advantageously be used as nuclear donorcells. By using cells which have histone hypomethylation the efficiencyof nuclear transfer may be increased.

A first aspect of the invention provides a method of producing an animalembryo, the method comprising transferring from a nuclear donor cellwhich has been selected on the basis that it is histone hypomethylatedat least a portion of the nuclear contents including at least theminimum chromosomal material able to support development into a suitablerecipient cell.

By a “cell which has been selected on the basis that it is histonehypomethylated” we include:

-   (i) testing a cell to determine if it is histone hypomethylated and    selecting the cell if it is found to be histone hypomethylated;-   (ii) experimentally determining that a first cell is histone    hypomethylated and selecting a second cell (the nuclear donor cell)    which is similar or identical to the first cell to thereby select a    histone hypomethylated cell to be used as a nuclear donor cell; and-   (iii) selecting a histone hypomethylated cell by selecting a cell of    a type which has been previously determined as being histone    hypomethylated (e.g. a resting B lymphocyte, preferably a small    resting B lymphocyte) or which has been previously determined as    being likely to be histone hypomethylated.

Preferably technique (ii) or (iii) is used.

With respect to technique (ii), by the second cell being “similar” tothe first cell we refer to the second cell being sufficiently similar tothe first cell (i.e. having a sufficient number of characteristics incommon) such that it is reasonable to infer that because the first cellexhibits histone hypomethylation the second cell also exhibits histonehypomethylation. Preferably, the first and second cells are from thesame population of cells, such as a population of cells which has beenenriched for histone hypomethylated cells.

By “experimentally determining that a first cell is histonehypomethylated and selecting a second cell (the nuclear donor cell)which is similar or identical to the first cell to thereby select ahistone hypomethylated cell to be used as a nuclear donor cell” weinclude determining that a single first cell is histone hypomethylatedand selecting a second cell which is similar or identical to the firstcell.

By “experimentally determining that a first cell is histonehypomethylated and selecting a second cell (the nuclear donor cell)which is similar or identical to the first cell to thereby select ahistone hypomethylated cell to be used as a nuclear donor cell” we alsoinclude determining that more than one first cell is histonehypomethylated and selecting a second cell which is similar or identicalto the first cells. The first cells may be subjected to the same ordifferent assay for histone hypomethylation. For instance, one firstcell or one aliquot of first cells may be tested with one type ofantibody and one second cell or one aliquot of second cells may betested with a second type of antibody etc.

With respect to technique (iii), by a type of cell which has beenpreviously determined as being likely to be histone hypomethylated weinclude a type of cell of which at least 70%, 80%, 90%, 95%, 99% or99.5% of cells of that type are hypomethylated.

Advantageously, the nuclear donor cell has reduced expression oractivity of one or more histone methyl transferases. Thus, in apreferred embodiment of the invention, the nuclear donor cell isselected on the basis that it has reduced expression or activity of oneor more histone methyl transferases.

Preferably, the nuclear donor cell has reduced expression or activity of1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the following enzymes capable ofmethylating lysine residues of histone H3 or histone H4: Suv39h1,Suv39h2, ESET, Ezh2, PR-set7, SET7/9, ASH1, ASH2, ALL1(trithorax), DOT1Land G9a.

By a “cell which has been selected on the basis that it has reducedexpression or activity of a histone methyl transferase” we include:

-   (i) testing a cell to determine if it is has reduced expression or    activity of a histone methyl transferase and selecting the cell if    it is found to have reduced histone methyl transferase expression or    activity;-   (ii) experimentally determining that a first cell has reduced    expression or activity of a histone methyl transferase and selecting    a second cell (the nuclear donor cell) which is similar or identical    to the first cell to thereby select a cell having reduced histone    methyl transferase expression or activity to be used as a nuclear    donor cell; and-   (iii) selecting a cell having reduced expression or activity of a    histone methyl transferase by selecting a cell of a type which has    been previously determined as having reduced expression or activity    of a histone methyl transferase or which has been previously    determined as being likely to have reduced expression or activity of    a histone methyl transferase.

Preferably technique (ii) or (iii) is used.

With respect to technique (ii), by the second cell being “similar” tothe first cell type we refer to the second cell being sufficientlysimilar to the first cell (i.e. having a sufficient number ofcharacteristics in common) such that it is reasonable to infer thatbecause the first cell has reduced expression or activity of a histonemethyl transferase the second cell also has reduced expression oractivity of a histone methyl transferase.

By “experimentally determining that a first cell has reduced expressionor activity of a histone methyl transferase and selecting a second cell(the nuclear donor cell) which is similar or identical to the firstcell” we include determining that a single first cell has reducedexpression or activity of a histone methyl transferase and selecting asecond cell which is similar or identical to the first cell.

By “experimentally determining that a first cell has reduced expressionor activity of a histone methyl transferase and selecting a second cell(the nuclear donor cell) which is similar or identical to the firstcell” we also include determining that more than one first cell hasreduced expression or activity of a histone methyl transferase andselecting a second cell which is similar or identical to the firstcells.

With respect to technique (iii), by a type of cell which has beenpreviously determined as being likely to have reduced expression oractivity of a histone methyl transferase we include a type of cell ofwhich at least 70%, 80%, 90%, 95%, 99% or 99.5% of cells of that typehave reduced expression or activity of a histone methyl transferase.

Persons skilled in the art will readily be able to devise assays fordetermining the level of expression or activity of histone methyltransferases. Immunofluorescence-based approaches or protein-basedtechnologies (ie. cells lysates and western blotting) may be used.

Expression of a histone methyl transferase may, for example, be assayedby using antibodies which detect the histone methyl transferase.Techniques for raising antibodies with desired specificities will bewell known to those skilled in the art. Moreover, some antibodies withappropriate specificities are commercially available.

Activity of a histone methyl transferase may, for example, be assayed byusing antibodies which detect methylated lysine residues. Techniques forraising antibodies with desired specificities will be well known tothose skilled in the art. Moreover, some antibodies with appropriatespecificities are commercially available. For example antibodies to 1×methyl H3-K9, methyl H3-K4 are available from Upstate Biotechnologies.

Histone methyl transferase activity can assessed by looking at theextent of incorporation of methyl groups into a specific histonesubstrate. This method has been published (for example ref 30, Kuzmichevet al., also the paper by Rea et al., 2000) and would be straightforwardfor someone skilled in the art.

Preferably, a “cell which has reduced expression or activity of ahistone methyl transferase” has ≦50% (and more preferably ≦45%, 40%,35%, 25%, 20%, 15%, 10%, 5%, 3%, 2% or 1%) of the average level ofhistone methyltransferase expression or activity of a population ofactivated or cycling cells of the same type (e.g. 24-hour activated,48-hour activated or 72-hour activated cells).

In one embodiment of the invention, a cell may be treated to reduce theactivity of a histone methyl transferase. In this way cells which aremore suitable for use as a nuclear donor cell may be obtained. In oneembodiment of the invention, the nuclear donor cell is a cell which hasbeen genetically engineered to have a reduced activity of one or morehistone methyl transferases. Preferably, a nuclear donor cell in whichone allele of the HMTase gene(s) in question has/have beendeleted/removed/inactivated (rather than both). Similarly, naturallyoccurring mutant cells having reduced activity of a histone methyltransferase may also be used.

Preferably, the nuclear donor cell is obtained by a method whichcomprises enriching a population of cells for suitable nuclear donorcells and selecting the nuclear donor cell from the enriched population.

Preferably, the enrichment process comprises separating histonehypomethylated cells from non-histone hypomethylated cells to therebyobtain a population enriched for histone hypomethylated cells.

Preferably, at least about 70%, 80%, 90%, 95%, 99%, or 99.5% of cells inthe enriched population are histone hypomethylated. More preferably,about 100% of cells in the enriched population are histonehypomethylated.

In one embodiment of the invention, the enrichment process comprisesseparating histone hypomethylated cells (e.g. small resting Blymphocytes) from histone hypomethylated cells having higher levels ofhistone methylation (e.g. large resting B lymphocytes). In this way apopulation of cells having particularly low levels of histonehypomethylation (such as small resting B lymphocytes), and which areparticularly suitable as nuclear donor cells, may be obtained.Preferably, at least about 70%, 80%, 90%, 95%, 99%, or 99.5% of theresulting cells are small resting B lymphocytes. More preferably, about100% of cells in the enriched population are small resting Blymphocytes.

In a particularly preferred embodiment of the invention, a population ofcells is enriched for resting B lymphocytes and a small resting Blymphocyte is selected from the population of cells enriched for restingB lymphocytes. A single small resting B lymphocyte may be selected or apopulation enriched for small resting B lymphocytes may be obtained fromthe population enriched for resting B lymphocytes. A small resting Blymphocyte may then be selected from the population of cells enrichedfor small resting B lymphocytes and be used as a nuclear donor cell.Preferably, at least about 70%, 80%, 90%, 95%, 99%, or 99.5% of thecells in the population enriched for small resting B lymphocytes aresmall resting B lymphocytes. More preferably, about 100% of cells in thepopulation enriched for small resting B lymphocytes are small resting Blymphocytes.

Various criteria may be used to obtain a population enriched forsuitable nuclear donor cells. For example, the cells may be separated onthe basis of one or more physical criteria, such as size or density, oron the basis of resistance to enzymatic digestion (for example in thecase of distinguishing hepatocytes and kupffer cells).

In a preferred embodiment, an enrichment step which differentiatesbetween small resting B lymphocytes and large resting B lymphocytes isperformed. Small and large B cells can be distinguished on the basis ofnuclear diameter (measuring at the widest place, for example as measuredby confocal microscopy). For example, in rodents such as mice, smallresting B cells have a nuclear diameter of about 8.0 μm or less(preferably ≦7.0 μm, 6.5 μm 6.0 μm, 5.5 μm, 5.0 μm, 4.5 μm, 4.0 μm, 3.5μm, 3.0 μm or 2.5 μm). Those skilled in the art will be able todetermine empirically those nuclear diameters which may be used tocharacterise B cells of other species into small and large resting Bcells. Similarly those skilled in the art will be able to determineempirically those nuclear diameters which may be used to characterise Tcells of rodents and other species into small and large resting T cells.

Separation of resting B cells into small and large resting B cells maybe done by density gradient separation, such as the method described byRatcliffe and Julius [22]. The population of resting B lymphocytes whichis separated into small and large resting B lymphocytes is preferablyobtained by a method comprising CD43-depletion of cells, such asCD43-depletion of splenic cells.

In one preferred embodiment, the nuclear donor cell is a lymphocyte(e.g. B lymphocyte or T lymphocyte) and the nuclear donor cell isobtained by a method which comprises enriching for small resting Blymphocytes (and/or small resting T lymphocytes) and selecting thenuclear donor cell from the population of small resting B lymphocytes(and/or small resting T lymphocytes).

In a one embodiment of the invention, an enrichment step whichdifferentiates between small resting T lymphocytes and large resting Tlymphocytes is performed.

A population enriched for suitable nuclear donor cells may be obtainedby enzymatic separation. In enzymatic separation, one or more enzymesare employed which digest unwanted cells. For example, kupffer cells maybe obtained from a liver cell suspension comprising kupffer cells andhepatocytes by incubation with pronase (see the Examples section).

Preferably, one or more cells from the enriched population (e.g.enriched for resting B lymphocytes or small resting B lymphocytes) aretested for histone hypomethylation.

Preferably, one or more cells from the enriched population (e.g.enriched for resting B lymphocytes or small resting B lymphocytes) aretested for reduced histone methyl transferase expression.

In one embodiment of the invention, the cells present in a tissuesection are tested for histone hypomethylation. In this manner, largenumbers of cells can be tested to identify cells which are histonehypomethylated which may be useful as nuclear donor cells. Thisinformation may then be used to select a histone hypomethylated nucleardonor cell. Thus, as described in the examples section,antibody-labelling of liver sections allowed the identification ofhistone hypomethylated kupffer cells. An enrichment step for kupffercells may then be performed (e.g. by enzymatic separation) and a nucleardonor cell selected from the resulting population of kupffer cells.

Histone hypomethylated cells may be readily distinguished frommethylated cells due to the considerable and readily-observabledifferences in their respective levels of histone methylation. Indeed,the level of methylation in hypomethylated cells appears to benegligible or absent (or at least undetectable). Thus, for practicalpurposes methylation may generally be considered to be an all or nothing(or almost nothing) event. Accordingly, the skilled person will readilybe able to appreciate whether a cell is hypomethylated or not.

Preferably, a cell is regarded as being histone hypomethylated ifhistone methylation is negligible or absent (absent being used herein tomean undetectable).

As demonstrated in the Example below, cells which are hypomethylatedinclude G₀ (resting) lymphocytes and some liver cells (as indicatedbelow these liver cells may be Kupffer cells). Also, the paternalgenome, early after fertilisation has been identified as beinghypomethylated (see Cowell et al. (2002)).

Cells which are not hypomethylated include activated lymphocytes, serumstarved fibroblasts and some post-mitotic cells (e.g. cumulus cells andmultinucleated muscle fibres).

Histone methylation may be assessed in various ways as outlined below.

The level of histone methylation can be assessed directly usingantibodies that detect methylated lysine residues. The level of antibodybinding is a direct reflection of the level of histone methylation ineach cell. Techniques for raising antibodies with desired specificitieswill be well known to those skilled in the art. Moreover, someantibodies with appropriate specificities are commercially available.For example antibodies to 1× methyl H3-K9, methyl H3-K4 are commerciallyavailable from Upstate Biotechnologies. See also the Materials andMethods section below.

To assess histone methylation, immunofluorescence-based approaches orprotein-based technologies (ie. cells lysates and western blotting) maybe used (see the Examples section below).

Histone methylation may be assessed with regard to one or more histonetypes. Preferably, the level of histone methylation is assessed withregard to H3 and/or H4, preferably with regard to H3.

The assessment of histone methylation may involve assessing whether oneor more histone residues are methylated. Obviously, for the assay to bemeaningful only methylation at histone residues which are known toundergo histone methylation is assessed.

With regard to techniques (ii) and (iii), in one embodiment it ispreferred that the level of histone methylation of said first cell or ofsaid cell type is assessed on the basis of methylation at one or moreresidues of H3.

Histone residues which may be methylated include lysine and arginineresidues. In one embodiment, methylation at one or more lysine residuesis assessed. In another embodiment methylation at one or more arginineresidues is assessed. Preferably, methylation at one or more lysineresidues and at one or more arginine residue is assessed.

Lysine residues of H3 which may be methylated in mammals includeresidues 4, 9, 27 and 36 (Rice and Allia (2001) Current Opinion in CellBiology, 13:263-273; and Richards and Elgin (2002) Cell 108, 489-500).Preferably, methylation at one, two, three or four of these lysineresidues is assessed. Preferably, methylation of H3^(K4) or H3^(K9) orH3^(K27) is assessed.

Preferably, methylation of both H3^(K4) and H3^(K9) is assessed.Preferably, methylation of H3^(K4), H3^(K9) and H3^(K27) is assessed.

Preferably, methylation at ≧two, three, four, five or six histoneresidues is assayed.

The assessment of histone methylation may involve assessing the extentof methylation (ie. mono-, di- or tri-methylation) at one or moreresidue(s).

It will be appreciated that an assessment of histone methylation mayinvolve assaying different histones for methylation and/or differenthistone residues for methylation and/or the extent of methylation atdifferent residues.

Preferably, a cell which is regarded as being histone hypomethylated hasnegligible or absent (i.e. undetectable) methylation at ≧one, two,three, four, five or six histone residues.

In one embodiment, a cell is regarded as being histone hypomethylated ifit has 10% or less (and more preferably ≦8%, 5% or 2%) of the level ofhistone methylation of one or more (and preferably any) of the celltypes listed above as examples of cells which are not hypomethylated.Preferably, a cell is regarded as being histone hypomethylated if it has10% or less (and more preferably ≦8%, 5% or 2%) of the level of histonemethylation of an activated or cycling lymphocyte.

Methods of quantifying histone methylation will be known to thoseskilled in the art or can be readily devised by those skilled in theart. For example, a semi-quantitative western blotting approach may beused.

In one embodiment of the invention, a cell is regarded as being histonehypomethylated if it has ≦50% (and more preferably ≦45%, 40%, 35%, 25%,20%, 15%, 10%, 5%, 3%, 2% or 1%) of the average level of histonemethylation of a population of activated or cycling cells of the sametype (e.g. 24-hour activated, 48-hour activated or 72-hour activatedcells).

The level of histone hypomethylation may be assessed as described hereinin relation to FIG. 7 a. Briefly, the level of histone methylation maybe assessed by comparing the cellular intensity of a mono- di- or tri-methylated H3-K9 labelled test cell with the average level of cellularintensity of a mono- di- or tri- methylated H3-K9 labelled population ofactivated (or cycling) cells. Following labeling of the cells withantibodies specific to mono- di- or tri- methylated H3-K9, confocalimages of the cells are taken at identical settings for each antibodystudied. The total labeling of the nucleus of each cell can then bequantitated calculating either the average pixel intensity of eachnucleus (pixel average) or the total intensity of each nucleus(integrated). The technique can be used to obtain a reading for anindividual “test” cell and this reading may then be compared with theaverage reading (preferably the mean of ≧about 25, 75, 150 cells)obtained for a number of “benchmark” cells which may be activated orcycling cells.

The term “embryo” as used herein includes all concepts of an animalembryo such as an oocyte, egg, zygote or an early embryo. Morespecifically, the term “embryo” used herein includes morulas (8-16cells), morulas (16-32 cells) and blastocysts (64 cells and above).

The term “nuclear donor cell” as used herein includes a cell from whichat least a portion of the nuclear contents including at least theminimum chromosomal material able to support development is transferredinto a suitable recipient cell. Similar expressions e.g. “nucleartransfer” should be interpreted in a likewise manner.

Preferably, the nuclear donor cell employed in the present invention isa mammalian cell. Preferably, the recipient cell is a mammalian cell.Preferably, the nuclear donor cell and the recipient cell are bothmammalian cells; preferably they are both ungulate, rat or murine cells.

In an alternative embodiment the nuclear donor cell and/or recipientcell is not a mammalian cell. The nuclear donor cell and/or recipientcell may, for example, be a Xenopus cell.

Preferably, donor cells and recipient cells from the same species areused. Preferably, the donor cell and recipient cell are both human cellsor mouse cells.

Cells derived from populations grown in vivo or in vitro and containing2n chromosomes (e.g. those in G0 or G1) or greater than 2n chromosomes(e.g., those in G2, which are normally 4n) may act as nuclear donorcells.

An example of an in vivo source of the 2n donor nucleus is a cumuluscell. One embodiment of the invention contemplates using donor nucleitaken from either in vivo or in vitro (i.e., cultured) sources of 2nadult somatic cells including, without limitation, epithelial cells,neural cells, epidermal cells, keratinocytes, hematopoietic cells,melanocytes, chondrocytes, B or T lymphocytes, macrophages, monocytes,nucleated erythrocytes, fibroblasts, Sertoli cells, cardiac musclecells, skeletal muscle cells, smooth muscle cells, and other cells fromorgans including, without limitation, skin, lung, pancreas, liver,kidney, urinary bladder, stomach, intestine, bone, and the like, andtheir progenitor cells where appropriate.

In one embodiment, the donor cell is a resting cell (G₀), preferably aresting B lymphocyte or resting T lymphocyte, preferably a small restingT lymphocyte. Preferably, the nuclear donor cell is a small resting Blymphocyte obtained or obtainable by density gradient separation, suchas described above or as in the Examples section.

In another embodiment of the invention, the donor adult somatic cell is“2-4C”; that is, it contains one to two times the diploid genomiccontent, as a result of replication during S phase of the cell cycle.This donor cell may be obtained from an in vivo or an in vitro source ofactively dividing cells including, but not limited to, epithelial cells,hematopoietic cells, epidermal cells, keratinocytes, fibroblasts, andthe like, and their progenitor cells where appropriate.

In one embodiment of the invention it is preferred that the donor cellis not selected from the group consisting of: a resting lymphocyte, aresting B lymphocyte, a liver cell, or a Kupffer cell.

Optionally the donor nucleus may be genetically modified. The donornucleus may contain one or more transgenes and the genetic modificationmay take place prior to nuclear transfer and embryo reconstitution. Sucha genetically modified donor nucleus may be used in the creation of atransgenic animal.

It should be noted that the term “transgenic”, in relation to animals,should not be taken to be limited to referring to animals containing intheir germ line one or more genes from another species, although manytransgenic animals will contain such a gene or genes. Rather, the termrefers more broadly to any animal whose germ line has been the subjectof technical intervention by recombinant DNA technology. So, forexample, an animal in whose germ line an endogenous gene has beendeleted, duplicated, activated or modified is a transgenic animal forthe purposes of this invention as much as an animal to whose germ linean exogenous DNA sequence has been added.

Preferably, the recipient cell is a one cell zygote, enucleated oocyte,embryonic stem (ES) cell or any other type of cell which may facilitatein the reprogramming of the donor nucleus. As will be appreciated frombelow, the recipient cell may be the “ultimate” recipient cell in whichcase the resulting embryo may directly give rise to a foetus or animal(offspring). Alternatively, in the case of serial nuclear transfer(discussed below), the recipient cell may not be the “ultimate”recipient cell and it may act as a nuclear donor cell.

Preferably, the enucleated oocyte is a mammalian enucleated oocyte.Enucleation may be achieved physically, by actual removal of thenucleus, pro-nuclei or metaphase plate (depending on the recipientcell), or functionally, such as by the application of ultravioletradiation or another enucleating influence.

Oocytes that may be used in the method of the invention include bothimmature (e.g., GV stage) and mature (i.e., Met II stage) oocytes.Mature oocytes may be obtained, for example, by inducing an animal tosuper-ovulate by injections of gonadotrophic or other hormones (forexample, sequential administration of equine and human chorionicgonadotrophins) and surgical harvesting of ova shortly after ovulation(e.g., 80-84 hours after the onset of estrous in the domestic cat, 72-96hours after the onset of estrous in the cow and 13-15 hours after theonset of estrous in the mouse). Where it is only possible to obtainimmature oocytes, they are cultured in a maturation-promoting mediumuntil they have progressed to Met II; this is known as in vitromaturation (“IVM”). Methods for IVM of immature bovine oocytes aredescribed in WO 98/07841, and for immature mouse oocytes in Eppig &Telfer (Methods in Enzymology 225, 77-84, Academic Press, 1993).

Preferably, the recipient cell to which the donor cell nucleus istransferred is an enucleated metaphase II oocyte, an enucleatedunactivated oocyte or an enucleated preactivated oocyte. At least wherethe recipient is an enucleated metaphase II oocyte, activation may takeplace at the time of transfer. Alternatively, at least where therecipient is an enucleated unactivated metaphase II oocyte, activationmay take place subsequently.

Once suitable donor and recipient cells have been selected, it isnecessary for the nuclear material of the former to be transferred tothe latter. The nuclear donor cell can be transferred intact into asuitable recipient cell, optionally with a broken cell membrane.Alternatively, the nuclear contents of the donor cell (or a portion ofthe nuclear contents including at least the minimum chromosomal materialable to support development) can be directly inserted into the cytoplasmof an enucleated oocyte.

Conveniently, nuclear transfer is effected by fusion. Three establishedmethods which have been used to induce fusion are: (i) exposure of cellsto fusion-promoting chemicals, such as polyethylene glycol; (ii) the useof inactivated virus, such as Sendai virus; and (iii) the use ofelectrical stimulation.

Alternatively, nuclear transfer is effected by microinjection.

Before or (preferably) after nuclear transfer (or, in some instances atleast, concomitantly with it), it is generally necessary to stimulatethe recipient cell into development by parthenogenetic activation, atleast if the cell is an oocyte. In one embodiment, the activation steptakes place from zero to about six hours after nuclear transfer in orderto allow the nucleus to be in contact with the cytoplasm of the oocytefor a period of time prior to activation of the oocyte. Activation maybe achieved by various means which will be well known to those skilledin the art.

There are several options for which the embryos made by the presentinvention may be used for.

In one embodiment, the embryo may be used in serial nuclear transfer.Thus, a second aspect of the invention provides a method of producing ananimal embryo, the method comprising transferring from a nuclear donorcell at least a portion of the nuclear contents including at least theminimum chromosomal material able to support development into a suitablerecipient cell wherein the nuclear donor cell is obtained from an embryoobtained by the method of the first aspect of the invention.

Preferably, the nuclear donor cell obtained from an embryo obtained inaccordance with the first aspect of the invention has been selected onthe basis that it is histone hypomethylated.

It will be appreciated that the embryo obtained by the method of thesecond aspect of the invention may be used for further rounds of serialnuclear transfer.

Preferably, an embryo obtained by the first or second aspects of theinvention is allowed to develop into a foetus or animal (i.e. liveoffspring). Thus, a third aspect of the present invention provides amethod of producing a foetus the method comprising allowing an embryoobtained by the first or second aspect of the invention to develop intoa foetus.

The step of allowing the embryo to develop may include the substep oftransferring the embryo to a female mammalian surrogate recipient,wherein the embryo develops into a viable foetus. The embryo may betransferred at any stage, including from the two-cell tomorula/blastocyst stage, as known to those skilled in the art.

A fourth aspect of the invention provides a method of producing anon-human animal the method comprising allowing an embryo obtained bythe first or second aspects of the invention or a foetus obtained by thethird aspect of the invention to develop into said non-human animal.

Those skilled in the art will appreciate that the cloned embryos of thepresent invention may be combined with fertilized embryos to producechimeric embryos, foetuses and/or offspring. Such chimeric embryos,foetuses and/or offspring are also included within the scope of thepresent invention.

In another aspect of the invention an embryo of the present invention isused in the preparation of an embryonic stem cell line. Thus, a fifthaspect of the present invention provides a method of producing anembryonic stem cell line, the method comprising transferring an embryoobtained by the method of the first or second aspect of the invention toa culture system.

A sixth aspect of the invention provides a method of producing anembryonic stem cell line, the method comprising isolating the inner cellmass of an embryo obtained by the method of the first or second aspectof the invention and transferring the inner cell mass to a culturesystem.

An embryonic cell line could find beneficial application in its use togenerate embryonic stem cells from a patient as a source of compatibleundifferentiated cells to be used in transplantation for the therapy ofdegenerative diseases.

In an seventh aspect of the invention, a cell could be treated toartificially reduce the level of histone methylation so as to render thecell histone hypomethylated. The cell could be employed as a nucleardonor cell in the above described methods of the present invention. Thetreatment may be chemical or enzymatic and may, for example, involvetreatment with a histone demethylase or with a histone methyltransferase(HMT) inhibitor¹.

An eighth aspect of the invention relates to the embryos, foetuses,non-human animals, and embryonic cells obtained by the methods describedabove.

A ninth aspect of the invention relates to the use of histonehypomethylation status as an indicator of the suitability of a cell toact as a nuclear donor cell. Histone hypomethylation status may beassessed as described above.

A tenth aspect of the invention provides a method of selecting a cell tobe used as a nuclear donor cell the method comprising selecting saidcell on the basis that it is histone hypomethylated.

An eleventh aspect of the invention relates to the use of a resting B orT lymphocyte or Kupfer cell as a nuclear donor cell. Preferably, theresting B or T lymphocyte is a small resting B or T lymphocyte.

Preferably, the small resting B lymphocyte is obtained by a methodcomprising selecting a small B lymphocyte from a population of cellscomprising resting B lymphocytes.

Preferably, the small resting B lymphocyte is obtained by a methodcomprising selecting a small B lymphocyte from a population of cellsenriched for resting B lymphocytes. Preferably, one or more of the cellsin the population of cells enriched for resting B lymphocytes are testedfor histone hypomethylation.

Preferably, one or more of the cells in the population of cells enrichedfor resting B lymphocytes are tested for reduced expression or activityof a histone methyl transferase. Preferably, at least about 70%, 80%,90%, 95%, 99%, or 99.5% of cells in the population enriched for restingB lymphocytes are histone hypomethylated. Preferably, at least about100% of cells in the population enriched for resting B lymphocytes areresting B lymphocytes.

In one embodiment, the population of cells enriched for resting Blymphocytes is obtained by CD43-depletion of splenic cells.

Preferably, the small B lymphocyte is obtained from the population ofcells enriched for resting B lymphocytes by visually detecting a smallresting B lymphocyte cell present in the enriched population andselecting the cell.

Alternatively, the small B lymphocyte is obtained from the population ofcells enriched for resting B lymphocytes by obtaining a population ofcells enriched for small resting B lymphocytes from the population ofcells enriched for resting B lymphocytes and selecting a small Blymphocyte from the population of cells enriched for small resting Blymphocytes.

Preferably, one or more of the cells in the population of cells enrichedfor small resting B lymphocytes are tested for histone hypomethylation.

Preferably, one or more of the cells in the population of cells enrichedfor small resting B lymphocytes are tested for reduced expression oractivity of a histone methyl transferase.

Preferably, at least about 70%, 80%, 90%, 95%, 99%, or 99.5% of cells inthe population enriched for small resting B lymphocytes are smallresting B lymphocytes. Preferably, at least about 100% of cells in thepopulation enriched for resting B lymphocytes are small resting Blymphocytes.

Preferably, the population of cells enriched for small resting Blymphocytes is obtained from the population of cells enriched forresting B lymphocytes by density gradient separation.

In principle, the invention is applicable to all animals, includingbirds, such as domestic fowl, amphibian species and fish species. Inpractice, however, it will generally be to placental mammals that thegreatest commercially useful applicability is presently envisaged. It iswith ungulates, particularly economically important ungulates such ascattle, sheep, goats, water buffalo, camels and pigs that the inventionis likely to be most useful, both as a means for cloning animals and asa means for generating transgenic or genetically modified animals. Itshould also be noted that the invention is also likely to be applicableto other economically important animal species such as, for example,horses, llamas or rodents e.g. rats, mice, rabbits and humans. However,due to ethical considerations, it may be desirable for certain aspectsof the invention not to be applied to humans.

The present invention will now be described by reference to theaccompanying Examples which are provided for the purposes ofillustration and are not to be construed as being limiting on thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. HP1β (M31) and Ikaros proteins are up-regulated andredistributed to constitutive heterochromatin in B lymphocytes followingmitotic stimulation. In (a) the kinetics of CD69 expression and BrdUincorporation by purified G₀ mouse B lymphocytes following mitoticstimulation with anti-IgM and CD40 antibodies is shown. Cells weresampled 0, 24, 48 and 72 hours (hrs) post-stimulation and the resultsshow representative histograms of CD45RA (B220), CD69 and anti-BrdUlabeling against cell number. In (b) the upper panels showrepresentative confocal images of the nucleus of B lymphocytessimultaneously labeled with anti-Ikaros and anti-HP1β (M31) at 0, 24 and72 hours post-stimulation. The nuclear periphery of each cells isoutlined by lamin B labeling. In the lower panels, confocal images oflymphocytes co-stained with CREST anti-sera and DAPI are shown forcomparison.

FIG. 2. Ikaros, HP1β Ezh2 and Bmi1 proteins are selectively up-regulatedin the nucleus of B lymphocytes following mitotic stimulation. Panel (a)shows western blots in which the abundance of specific proteins withincytoplasmic (CE), soluble and insoluble nuclear extracts (NE-s and NE-i,respectively) are compared at different times after B lymphocyteactivation. In panel (b) representative confocal images of thedistribution of Ezh2, Eed, Bmi1 and ESET proteins relative to PI-labeledin the nuclei of quiescent (0 hrs) and cycling (72 hrs) B lymphocytesare shown.

FIG. 3. Selective increase in histone methylation in B lymphocytesfollowing mitotic stimulation. Panel (a) shows the distribution ofmethylated H3-K9, H3-K4, H3-K27, acetylated H3-K9, H3-K14 or H4 inquiescent (0 hrs) and cycling (72 hrs) B cells measured byimmunofluorescence, relative to DAPI labeling. Panel (b) shows therelative abundance of these modified histones estimated by westernblotting of protein lysates harvested 0, 24 and 72 hrs after lymphocytestimulation.

FIG. 4. H3-K9 methylation in resting and cycling B lymphocytes. In (a)the distribution of mono- (Me) di- ((Me)₂) and tri- ((Me)₃) methylatedhistone H3 in quiescent (0 hrs) and cycling (72 hrs) B cells is shownrelative to DAPI labeling. Panel (b) shows the relative distribution oftri-methyl H3-K9, HP1β and DAPI labeling in resting (0 hrs) and cycling(72 hrs) B cells.

FIG. 5. Hypomethylation of histone H3 in liver Kupffer cells. In (a)mouse adult liver sections labeled with 4xmethyl H3-K9 and DAPI, apopulation of cells lacking methylated H3-K9 were seen (arrowed). Panel(b) shows labeling of isolated liver cell suspensions with biotinylatedCD45 antibody revealed with avidin FITC either alone (left) orco-staining with antibody to methylated H3-K9 (4xmethyl H3-K9) ormethylated H3-K4. Panel (c) shows methylated H3-K9 labeling (4xmethylH3-K9) of freshly isolated Kupffer cells (0 hours) and following mitoticstimulation (24 hours in GM-CSF and IL-3)(upper panels), where Kupffercells were identified by co-labeling with anti-CD45 (lower panels).

FIG. 6: Histone H3-K9 methylation is absent (or low) in quiescent mouseB lymphocytes and dynamically up-regulated upon mitotic stimulation.Methylated H3-K9 in the nucleus of G₀ (0 hrs) and cycling (72 hrs) Blymphocytes is shown (4xmethyl H3-K9 labeling) relative to DAPI-intenseregions in the nucleus of cells isolated from normal male wild type) andSuv39h-deficient (left and right-hand columns, respectively). Thedistribution of Ikaros proteins in the nucleus of cycling B lymphocyteswas not affected by the absence of Suv39h HMTases, as shown in the lowerpanels.

FIG. 7: Cellular intensities of mono- di- and tri- methylated H3-K9labeling of resting and activated B cells. Following labeling of resting(day 0), 24 hour activated (day 1) and 72 hour activated B cells (day 3)with antibodies specific to mono- di- and tri-methylated H3-K9 (panela). Confocal images of between 300-400 cells were taken at identicalsettings for each antibody studied. The total labeling of the nucleus ofeach cell was then quantitated calculating either the average pixelintensity of each nucleus (pixel average) or the total intensity of eachnucleus (integrated). Individual events were then grouped into discretequanta of intensities and expressed in histograms of increasing quantaof intensities (x axis) against cell number (y axis). The nucleardensity of labeling of tri-methylated H3-K9 appeared low as labeling wasconcentrated to discrete foci. To allow for this, the proportion ofcells that showed focussed H3-K9 labeling at each time point wasassessed and is shown beneath the representative images shown in (b).

FIG. 8: Reduced H3-K9 methylation in resting versus cycling Tlymphocytes Lymphocytes were isolated from the lymph nodes of mice (0hrs) and T cells were stimulated by incubation on culture dishes coatedwith anti-TCR β chain antibody (H57, Pharmingen), in media supplementedwith anti-CD28 (Pharmingen) and IL-2 for 3 days (72 hrs). T cells inthese populations were identified by surface staining with APC-coupledanti-TCR β chain (H57, Pharmingen), and the samples were then fixed with2% paraformaldehyde and IF analysis performed using antibody specificfor M31/HP1β, and DAPI. The images show four cells, three of which arepositive for surface T cell receptor (TCR) and can therefore be clearlyidentified as T cells. In resting T cells (left panels) an absence ofM31 at pericentric heterochromatin (visualised by intense DAPI label) isapparent. However, in activated T cells M31 is redistributed topericentric heterochromatin and co-localises with DAPI-bright areas.

FIG. 9: Reversal of transgene silencing is more efficient using donornuclei from G₀ cells than activated B cells. Nuclei from resting (0-24hours) or active (48-72 hours) B cells carrying the silent EGFPtransgene were transferred into fertilised embryos 18-21 hours postinjection with human chorionic gonadotrophin (BL6/D2×BL6/D2). Embryossurviving the transfer procedure were cultured overnight in M16 media(Specialty Media). On day one after transfer, 2-cell embryos weretransferred into glucose-supplemented CZB media. The majority ofoperated embryos reached morulae or early blastocyst stage but weresomewhat developmentally retarded as compared to control embryos. On dayfour after transfer, GFP expression was assessed. Consistently, twice asmany embryos showed strong GFP expression after transfer with resting Bcell nuclei as compared with activated B cells. Data from variousexperiments are shown.

FIG. 10: Heterogeneity of methylation patterns in resting B cells.Increased amounts of methylation were observed in large resting B cellsas compared with small resting B cells.

FIG. 11: Peptide-blot analysis determining the specificity of antibodiesused in this study. Peptides representing histone H3N-termini eithermono-, di-, or trimethylated at the indicated position were transferredonto nitrocellulose in quantities of either 50, 10 or 2 pmoles. Theseblots were probed with the respective antibodies at the indicatedconcentrations. Binding efficiency was finally determined by a stainingreaction of a secondary peroxidase coupled antibody (Jackson ImmunoResearch Laboratories).

Four antibodies raised against a 2×-branched peptide and one raisedagainst a 4×-branched peptide were generated within the group of ThomasJenuwein (left panel), whereas the two antibodies raised against alinear peptide are commercially available from Upstate Biotechnologies(UBI) (right panel). Both the UBI “linear” α-dimethyl H3-K9 antibody andthe “4×-branched” α-dimethyl H3-K9 antibody have a major reactivityagainst methylated lysine 9 of the histone H3 tail but also havereactivity against methylated lysines 4 and 27 of histone H3. The UBI“linear” α-dimethyl H3-K4 antibody and “2×-branched” α-mono, di- andtri-methyl H3-K9 antibodies and the α-tri-methyl H3-K27 antibody displayspecific binding activity.

Abbreviations

-   BrdU Bromodeoxyuridine-   CE Cytoplasmic extract-   ChIP Chromatin Immunoprecipitation-   DAPI 4′,6-Diamidino-2-phenylindole-   HMTases Histone methyl transferases-   HP1 Heterochromatin protein 1-   IgM Immunoglobulin M-   IL-3 Interleukin 3-   IL-4 Interleukin 4-   IF Immunofluorescence-   Me Mono-methyl-   (Me)₂ Di-methyl-   (Me)₃ Tri-methyl-   NE-i Nuclear extract insoluble-   NE-s Nuclear extract soluble-   PcG Polycomb group-   PEV Position Effect Variegation-   PRC1 Polycomb repressive complex 1-   TCR T cell receptor-   TrxG Trithorax group

EXAMPLES SUMMARY BACKGROUND

Covalent modification of histones has been proposed as a possiblemechanism of epigenetic inheritance based on observations that differentpatterns of histone methylation and acetylation are predictablyassociated with distinct chromatin and transcriptional states. Toinvestigate their role in transcriptional memory, the extent of histoneH3 and H4 modification in quiescent (G₀) and actively cycling mouse Blymphocytes was examined.

Results

We observed a generalised reduction in histone H3 methylation at lysineresidues 4 (H3-K4), 9 (H3-K9) and 27 (H3-K27) in purified G₀ splenic Bcells and the absence of heterochromatin-associated proteins HP1β andIkaros at centromeric heterochromatin. Mitogenic stimulation resulted ina rapid increase in methylation at all three histone H3 residues priorto the onset of DNA replication, coincident with an up-regulation andglobal redistribution of Polycomb group proteins Bmi1, HP1 and of theEzh2 and ESET MTases. Histone hypomethylation was also evident amongnon-cycling populations of Kupffer cells (but not hepatocytes) in adultliver and was reinstated following mitotic stimulation.

Conclusions

These results suggest that global methylation of histone H3 is moredynamic than had been previously appreciated and that histonehypomethylation is a feature of specific G₀ populations in vivo.

Introduction to Experimental Work

Here we investigate the contribution of histone modifications toepigenetic memory by comparing the extent of histone acetylation andmethylation between purified resting (G₀) and cycling B lymphocytes. Therationale for this comparison lies with the capacity of quiescentlymphocytes to survive for extensive periods in vivo, but only re-enterthe cell cycle upon antigenic stimulation. This implies that epigeneticinformation that defines both the lineage and developmental stage ofdifferentiated B cells is actively retained in long-term quiescentcells. Consistent with this assumption, it is noteworthy that lymphocyteproliferation is severely impaired in mice lacking several individualPcG proteins [18-20]. We have previously shown that quiescent Blymphocytes lack some features found in cycling cells, most noticeably alack of spatial association of transcriptionally inactive genes andIkaros proteins at pericentric heterochromatin [21]. Here we directlycompared histone modifications between cycling and non-cyclinglymphocytes in order to assess the role of this putative ‘code’ inconveying cellular memory. Surprisingly, levels of H3-K4, H3-K9, H3-K27methylation and Ezh2 and ESET HMTases were reduced or not detectable inquiescent primary B cells. These data show that chromatin compositiondiffers significantly between resting and cycling cells and suggest thathistone methylation is not necessarily a stable epigenetic imprint.

Materials and Methods

Purification and Activation of Resting B Lymphocytes from Spleen

Resting B Cell Purification from Spleen

Spleens of young (6-10 week old) mice were dissected and minced to yieldsingle cell suspensions. Erythrocytes in this population were removed bytreatment with Geyes solution (to lyse erythrocytes). Geyes solution wasprepared by mixing 20 parts stock solution A (650 mM NH₄Cl, 25 mM KCl, 4mM Na₂HPO₄.12H₂O, 1 mM KH₂PO₄, 28 mM Glucose) to 5 parts stock solutionB (20 mM MgCl₂.6H₂O, 6 mM MgSO₄.7H₂O, 30 mM CaCl₂) to 5 parts stocksolution C (267 mM NaHCO₃) to 70 parts sterile distilled water. To lyseerythrocytes, single cell suspensions were mixed with Geyes solution ina 1:4 ratio, and held on ice for 2 minutes before washing in media.

To remove CD43-positive cells from the cell suspension, cells werewashed with cold (4° C.) buffer (0.5% BSA in PBS A) cells were incubatedwith anti-CD43 (Ly-48)-coupled micro beads (Miltenyi Biotech) in bufferaccording to manufacturers' instructions. Labelled (CD43-posistive)cells were washed in buffer and passed through a magnetised depletioncolumn (Miltenyi Biotech). The column retains all paramagenticallylabelled cells but allows unlabelled (CD43-negative) cells to passthrough.

Where stated the CD43 negative B cells were enriched by density gradientseparation [21]. Where density gradient separation was used,CD43-negative cells were separated on a discontinuous Percoll gradientprepared and utilised as described previously (Ratcliffe and Julius,1983). Briefly CD43-negative cells were applied to a discontinuousPercoll gradient (prepared with density steps 1.060, 1.079, 1.085, 1.092and 1.109 g/ml) and small resting B cells were recovered at the1.079-1.085 g/ml density interface following centrifugation (30 minutesat 1500 g). In this way small resting B cells could be prepared. B cellactivation was induced by culturing cells in IMDM media containing 10%fetal bovine serum (Sigma) and antibiotics and 20 μg/ml purifiedanti-CD40 (monoclonal antibody FGK45), 101 g/ml purified anti IgM(monoclonal antibody H3074) and 2% IL-4 containing supernatant (from aT-helper cell line). Fluorescein-labeled antibodies to B220 and CD69 (BDPharmingen) were used for FACs analysis to verify the phenotype andactivation status of cells.

BrdU incorporation studies were performed using ex vivo resting mature Bcells. Cells were cultured in media containing 50 μM BrdU with eitherIL-4 for 24 hours (for un-stimulated cells) or following activationusing anti-IgM, anti-CD40 and IL-4 as described above. BrdUincorporation was revealed as previously described [54]; cells werefixed in ice cold 70% EtOH overnight at 4° C., washed in ice cold PBS,denatured in 3M HCl with 0.5% Tween for 20 min, followed by incubationin 0.01M sodium tetraborate solution for 3 min. After washing (2× in icecold PBS) the cells were incubated in FITC-conjugated anti-BrdUmonoclonal antibody (BD Pharmingen) before being washed and analysed byflow cytometry using a FACScan (Becton Dickinson).

Preparation of Liver Sections and Cell Suspensions

Liver sections were prepared and labeled as outlined elsewhere. Livercells were prepared using a two-step Procedure previously described(Seglen, 1972, Exptl Cell Res 74 p 450; Seglen, 1998 Cell Biology: alaboratory handbook, Volume 1, p 119) with minor modifications. Wholeliver was isolated from a recently sacrificed mouse, washed and a 25 gneedle was inserted into the vena cava. Blood was rinsed from the liverby continuous perfusion with a large volume of pre-perfusion buffer (0.5mM EGTA, 0.142M NaCl, 0.007M KCl, 0.01M HEPES, pH 7.4) until the tissueassumed a light tanned appearance. Pre-warmed (37° C.) collagenasebuffer (0.5 mg/ml Collagenase (Sigma), 0.067M NaCl, 0.007M KCl, 0.005MCaCl₂.2H2O, 0.1M HEPES, pH 7.6) was then introduced through the venacava continuously for 5-10 minutes until the structure of the tissuebegan to disintegrate. The tissue remnants were transferred into freshmedia and single cells were liberated by gentle agitation. The resultingcell suspension (which contains hepatocytes and Kupffer cells) waspassed through a 25 g needle washed twice in chilled media.

Isolation of non-parenchymal (kupffer cells) from liver cell suspensionscan be achieved either by pronase digestion or by density gradientseparation.

Pronase Digestion of Liver Cell Suspensions

Liver cell suspensions were incubated with 0.1% pronase (Sigma) for 1hour at 37° C. (as per Seglen P O: Preparation of isolated rat livercells. In: Methods in Cell Biology. pp. 29-83; 1976: 29-83.p 74) whichdigests/kills hepatocytes (cellular debris was removed by repeatedwashing and centrifugation). As this method can induce the activation ofnon-parenchymal cells an alternative (non-enzymatic) method ispreferred.

Density Gradient Enrichment of Kupffer Cells from Liver Cell Suspensions

As non-parenchymal cells are less dense than most parenchymal cells,these can be separated on a discontinuous metrizamide (or percoll)gradient (an example is given in Seglen P O: Preparation of isolated ratliver cells. In: Methods in Cell Biology. 1976. FIG. 23 (page 77). Cellsuspension were centrifuged above a 15% buffered metrizamide cushion(density 1.08 gm/cm³) for 60 minutes at 3500 rpm (see FIGS. 17 and 23 inSeglen P O: Preparation of isolated rat liver cells. In: Methods in CellBiology.).

Kupffer Cell Activation (to Restore Histone Methylation)

Freshly isolated Kupffer cells were activated by overnight incubation inIMDM media containing 10% Fetal bovine serum, antibiotics, 5% WEHI-3Bsupernatant (containing IL-3) 10 ng/ml murine GM-SCF and 10 ng/ml murineCSF-1.

Antibody Labeling and Fluorescence Microscopy

Antisera used for immunofluorescence and western blotting studies were;anti N- and C-terminus Ikaros [55], anti HP1β/M31 (Serotec) [24], antilamin B (Santa Cruz), human CREST autoimmune sera, anti-4xmethyl H3-K9,anti mono-methyl H3-K9, anti di-methyl H3-K9, anti tri-methyl H3-K9 andanti methyl H3-K27 [34, 36, 39] anti 1× methyl H3-K9, anti methyl H3-K4,anti acetyl H3-K9, anti acetyl H3-K14 (from Upstate Biotechnologies),anti pan-acetyl H4 (Serotec), anti Enx1 [56], anti EED [57], anti BMI1[58]. Additional control antibodies used in these analyses were antiHP1β (Euromedex), anti ORC1 (Serotec) and anti PCNA (Sigma).

For IF labeling, cells were attached to glass coverslips pre-coated withpoly-L-lysine, washed in PBS and fixed in 2% paraformaldehyde for 10min. The samples were washed in PBS and quenched with 0.05M NH₄Cl in PBSfor 5 min, before further washing in PBS and permeabilisation with 0.3%Triton in PBS for 5 min. Samples were incubated sequentially in blockingsolution (0.2% Fish gelatin (Sigma) in PBS) for 30 min and primaryantisera (diluted appropriately in blocking buffer with 5% normal goatserum) for 1 hour in a humid chamber. Following washing in blockingsolution, samples were incubated for a further 30 min influorochrome-labeled secondary antibody (diluted appropriately inblocking buffer and 5% normal goat serum). Slides were washed twice (3min/wash) in wash buffer, once in PBS alone and mounted in Vectashield(Vector) supplemented with DAPI (0.1 μg/ml). Where goat primaryantibodies were utilized, fetal calf serum replaced normal goat serum asa blocking reagent.

Immunofluorescence staining of histone modifications was performed asdescribed [36] with minor modifications. Cells were attached to glasscoverslips pre-coated in poly-L-lysine and washed in PBS. Samples werefixed in 2% paraformaldehyde for 10 min, washed in PBS and thenincubated in wash buffer for 5 min (wash buffer; PBS, 0.2% BSA, 0.1%Tween20). Preparations were then sequentially incubated, in a humidchamber, in blocking solution (blocking solution; PBS, 10% normal goatserum, 2.5% BSA, 0.1% Tween20) for 30 min, primary rabbit antisera(diluted in blocking solution) for 1 hour and Alexa 488 conjugated goatanti-rabbit IgG(H+L) diluted appropriately in blocking buffer. Sampleswere mounted in Vectashield supplemented with DAPI and visualized eitherby confocal microscopy using a TCS-SP1 (Leica Microsystems) or using anAxioplan 2E microscope (Zeiss), Metamorph 4.0 software and images wereprocessed using Adobe photoshop 6.0.

Preparation of Nuclear Extracts from Ex Vivo B Lymphocytes and WesternBlot Analyses

For preparing NE-i, NE-s and cytoplasmic (CE) extracts, B cells werewashed in ice cold PBS, centrifuged at 600 g for 4 min in a chilledcentrifuge (4° C.) and resuspended in ice cold nuclei lysis buffer (10mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA,supplemented with protease inhibitor cocktail and phosphatase cocktail(Sigma) and 1 mM DTT). Lysis buffer containing 0.75% NP40 was addeddrop-wise until the concentration of NP40 reached 0.15% and then left onice for 2 min before centrifugation at 400 g for 2 min. Thenon-chromatin cytoplasmic fraction (supernatant) was collected and theremaining nuclei were washed once in lysis buffer and centrifuged againas previously. Chromatin was solubilised by DNA digestion with 1 mg/mlof RNAse-free DNAse I (Sigma) in lysis buffer for 30 min at 30° C.NH₄(SO₄) was added from a 1 M stock solution in lysis buffer to a finalconcentration of 0.25 M. After 5 min on ice, samples were pelleted bycentrifuging at 1500 g for 3 min and DNAse I soluble material collected.The pellet of DNAse I insoluble material was then solubilised in Ureabuffer (8M urea, 0.1M NaH₂PO₄, 0.01M Tris-HCl pH 8.0) and the proteinextracts were quantified and stored at −70° C.

Histone proteins were isolated from whole cells by acid extraction.1×10⁷ B cells were pelleted and resuspended in 1 ml PBS (4° C.) andcentrifuged (500 g for 5 min) and the supernatant removed. Cell pelletswere resuspended in 180 μl of ice cold lysis buffer (10 mM HEPES pH 7.9,1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT and 1.5 mM PMSF), 20 μl of 2M HCladded and incubated on ice for 30 min. Following acid lysis the solutionwas centrifuged 11000 g for 10 min at 4° C., the supernatant of acidsoluble proteins collected and sequentially dialyzed against 0.1M aceticacid (twice for 1 hour) and water (1 hour, 3 hours and overnightrespectively). The protein solution was quantified and stored at −70° C.Western blotting of protein extracts was carried out as describedpreviously [21].

Results

Global Changes in Chromatin as Lymphocytes Enter the Cell Cycle

Resting (G₀) B lymphocytes can be purified from the spleen andstimulated to enter the cell cycle and generate progeny in which thecorrect lineage affiliation and developmental stage is faithfullytransmitted. Non-cycling B220⁺ B lymphocytes were isolated from thespleens of normal mice (by CD43 depletion and density gradientseparation [22]) and stimulated with anti-IgM, and anti-CD40 in thepresence of interleukin-4 (IL-4). Under these conditions cells expressthe activation marker CD69 within 24 hours and begin DNA synthesis, asdetected by BrdU incorporation, 48 to 72 hours after stimulation (FIG. 1a). The distribution of heterochromatin-associated proteins (Ikaros,HP1β and CENP-A) in quiescent and activated cells was monitored byimmunofluorescence (IF) and confocal microscopy in which all microscopesettings and the laser power were kept constant so that the relativeabundance and distribution of proteins could be directly compared (FIG.1 b). In purified resting B cells, Ikaros protein was low or absent butincreased following activation and re-located to centromeric domains asreported previously [21]. Low levels of HP1β/M31 were detected in thenuclei of resting B cells. These increased slightly over 24 hours, butHP1β/M31 did not localise to DAPI-intense centromere ‘clusters’ until48-72 hours after activation. At this time, as lymphocytes began celldivision, HP1β/M31 and Ikaros proteins co-localised around centromericDNA as previously reported [23, 24]. This kinetic re-distribution ofIkaros and HP1β/M31 proteins was confirmed using antibodies specific foralternative regions of these proteins (not shown). This, together withthe demonstration that CREST antisera detected centromeres throughout Bcell activation (FIG. 1 b, lower panels) rules out the possibility thattechnical problems such as epitope masking or restricted antibodyaccessibility account for a lack of Ikaros and HP1β detection atconstitutive heterochromatin domains in G₀ lymphocytes.

Differences in chromatin composition between resting and activated Bcells were also confirmed by western blotting. Nuclei were isolated fromresting and activated cells by partial NP40 lysis, a treatment thatresults in the removal of non-chromatin-bound proteins. Extracts werethen subjected to DNase I digestion and soluble (NE-s) or insoluble(NE-i) nuclear fractions were derived and analysed by SDS-PAGE andwestern blotting (FIG. 2 a). Controls included PCNA (a component of theDNA replication machinery synthesized as cells enter S-phase) and ORC1(a protein which marks origins of replication in quiescent and cyclingcells, used here to estimate the equivalence of protein loading). Lowlevels of PCNA were detected in samples 48 hours after stimulation,becoming more abundant in chromatin fractions after 72 hours. Thisobservation is consistent with most lymphocytes entering S-phase at thistime and mirrors the kinetics of BrdU incorporation shown in previousanalyses (FIG. 1)[21]. Ikaros proteins corresponding to the majorisoforms present in lymphocytes (isoforms I, II, [25]), were absent fromG₀ samples (0 hours), but were seen to accumulate in NE-i fractions48-72 hours after stimulation (FIG. 2 a). HP1β/M31 protein was presentin the soluble chromatin compartment (NE-s) throughout B cell activationbut showed a progressive recruitment to insoluble fractions (NS-i)following activation.

Binding of HP1β to pericentric heterochromatin has previously been shownto depend on the Suv39h histone methyltransferases [26, 27]. The SETdomains of Suv39h1 and Suv39h2 catalyse methylation of H3-K9 and providea high affinity binding site for M31/HP1β [26, 27]. An analogousmechanism probably operates in the recruitment of the Polycomb RepressorComplex-1 (PRC1) to other genomic sites; PRC1 recruitment followsmethylation of H3-K9 and H3-K27 by a separate PcG complex that containsthe SET domain protein Ezh2, Eed and histone deacetylases [28-31]. Inview of these findings we examined the distribution of severaladditional PcG and HMTase proteins in quiescent and cycling B cells(FIGS. 2 a and 2 b). Ezh2 and the PRC1-component Bmi1 were selectivelyupregulated following B cell activation. Both proteins were detected inchromatin-bound and soluble nuclear fractions and their abundanceincreased following lymphocyte activation (FIG. 2 a). In contrast, Eedlevels (detected by an antibody that recognizes both putative proteinsencoded by two alternatively transcribed mRNAs, [32]) remainedrelatively unchanged. The selective up-regulation of Ezh2 and Bmi1 PcGproteins was confirmed by IF labeling. Ezh2 staining was low in restingcells but increased in the nucleus of actively proliferating cells (FIG.2 b, 72 hours top panel). Small nuclear foci of Bmi1 were evident insome resting B cells and the intensity and number of nuclear fociincreased dramatically upon cell activation. This contrasted with thebroadly equivalent nuclear distribution of Eed protein in resting andactivated cells. Expression of a ESET, a second SET domain-containingHMTase, also increased following B cell activation and was evident atnon-heterochromatic foci (non DNA-dense regions) within the nucleus(FIG. 2 b).

H3 Methylation is Reduced in Quiescent B Cells Isolated Ex Vivo

The redistribution of HP1β/M31, Ikaros, Ezh2, Bmi1 and ESET proteins inB cells following mitogenic stimulation parallels the reported nuclearredistribution of genes in response to the activation of quiescentlymphocytes and fibroblasts [21, 33]. One possible explanation for theredistribution of these proteins could be underlying changes in histonemethylation. To assess this possibility, the extent of H3-K9 methylationin resting and activated B lymphocytes was assessed by IF and westernblotting using anti-methyl H3-K9 antibodies raised against either abranched peptide containing four di-methylated H3-K9 termini(α-4×-di-methyl H3-K9 or 4× methyl), or a single di-methylated H3-K9terminal peptide (α-di-methyl H3-K9 or methyl H3-K9). The α-4×-di-methylH3-K9 antibody primarily detects di- and tri-methylated H3-K9 but alsohas some reactivity against H3-K27 and labels pericentricheterochromatin and some euchromatic sites, whereas the α-di-methylH3-K9 primarily detects di-methylated H3-K9 (FIG. 11) [34]. Di- andtri-methylated H3-K9 was barely detected in quiescent lymphocytes by IF(FIG. 3 a) consistent with very low levels found by western blotting(FIG. 3 b). This was surprising since H3-K9 methylation has long beenconsidered a robust modification, which on the basis of the underlyingbiochemistry has been thought to be almost permanent in nature [1, 35].Following activation for 72 hours, di- and tri-methylated H3-K9 hadbecome highly abundant within the nucleus of activated lymphocytes (FIG.3 a) and was focussed around DAPI-dense regions, consistent withprevious reports [36]. Using a panel of antibodies that recognisealternative lysine residues, methylation of H3-K4, H3-K9, and H3-K27 waslow or undetectable in quiescent B cells but substantially increased incells preparing for division, being routinely detected within 24 hoursof activation (FIGS. 3 a and b). These data suggest a global reductionin histone methylation in resting B cells in both euchromatic andheterochromatic regions of the genome. In contrast to methylation,acetylation of H3-K9, H3-K14 and H4 appeared broadly similar in restingand activated B cells (FIG. 3 a, lower panels and FIG. 3 b, right-handpanel). In particular, acetylated H3-K9, H3-K14 and H4 were readilydetected in quiescent B lymphocytes. These data show that whereashistone acetylation is robustly retained by quiescent cells, histonemethylation appears to be a less stable epigenetic imprint inlymphocytes.

To investigate whether increases in H3-K9 methylation areSuv39h-dependent, we examined B cells from mice lacking both Suv39h1 andSuv39h2 HMTases. B cells from these mice (Suv39h−/−) mice showed asignificant increase in euchromatic H3-K9 methylation upon activation,but in contrast to normal cells, no enrichment ofheterochromatin-associated H3-K9 methylation (around DAPI intenseregions) was evident [36]. Interestingly, Ikaros proteins were focussedat centromeric heterochromatin in activated Suv39h−/− lymphocytes, inthe absence of local HP1β/M31 accumulation. This demonstrates thatIkaros binding to pericentric regions is independent of HP1, compatiblewith recent evidence that whereas HP1 interacts with lysine 9 methylatedH3 proteins [26, 27, 37], Ikaros binds directly to repetitive DNAsequences that flank centromeres [38]. These data indicate that the highlevels of methylated H3-K9 that typically surround the centromeres ofinterphase and metaphase chromosomes are not in fact constitutive in Blymphocytes, but are acquired by cells upon entry into cell cycle.

Global Up-Regulation of H3-K9 Methylation in Activated B Lymphocytes

To examine the selectivity of H3-K9 methylation upon B cell activation,antibodies capable of discriminating between the three different statesof H3-K9 methylation (mono-, di- or tri-methylated lysine 9) were usedto examine quiescent and cycling cells (see FIG. 11). Antibodies used inprevious analysis (α-4x-di-methyl H3-K9 and α-di-methyl H3-K9)preferentially recognise di/tri and di methyl H3-K9, respectively, butare inefficient at detecting the mono-methylated state. Using antiseraspecific for one (Me), two (Me)₂ or three (Me)₃ methyl groups at H3-K9[39] we consistently observed very low labeling of quiescent B cells,which increased markedly upon activation (FIG. 4 a). This was quantifiedby calculating the average pixel labeling intensity (pixel average) ofeach nucleus examined or the total intensity of each nucleus(integrated) 0, 1 and 3 days after stimulation (FIG. 7). Tri-methylH3-K9 labeling was observed only after mitotic stimulation and wasconfined to discrete locations within the nucleus coincident withDAPI-bright, condensed DNA domains. Confirmation that tri-methyl H3-K9(FIG. 4 b) localised at constitutive heterochromatin in cyclinglymphocytes was obtained by co-staining with antibody to HP1β (FIG. 4b). In cycling B cells tri-methyl H3-K9 and HP1β domains routinelyco-localised with DAPI-intense regions, an observation that isconsistent with reports that HP1β recognises tri-methyl H3-K9 [39].

Hypomethylation of Kupffer Cells in Mouse Liver

To determine whether reduced histone methylation was typical of other G₀cell types we also examined ex vivo T lymphocytes and non-cycling cellswithin the liver. Resting lymph node T cells, identified by T cellreceptor (TCR) expression, showed low levels of histone methylation,particularly tri-methylated H3-K9 as recognized by HP1β (see FIG. 8).Liver sections labeled with α-4×-di-methyl H3-K9 (4xmethyl H3-K9) showedevidence of two distinct cell populations. The majority of cells hadlarge nuclei (12-16 μM diameter) and expressed high levels of histonemethylation. A second population with smaller nuclei (8-9 μM diameter)lacked H3-K9 methylation (FIG. 5 a). The relative abundance of the twocell types was consistent with most cells being hepatocytes and theminority of smaller cells being Kupffer cells. To confirm this weprepared single cell suspensions of murine liver by collagenasetreatment using established protocols [40], and identified Kupffer cellson the basis of expression of the leukocyte-specific membrane proteinCD45. As shown in FIG. 5 b, Kupffer cells expressing surface CD45(identified by biotinylated anti-CD45 and FITC-avidin) were convenientlydiscriminated from larger hepatocytes in which endogenous biotin wasrestricted to the cytoplasm. Co-labeling of liver cell suspensions with4xmethyl H3-K9 confirmed that the hepatocytes showed high levels ofH3-K9 di/tri-methylation while no labeling was apparent in the nuclei ofKupffer cells. Methylated H3-K4 was also detected in hepatocytes but notin Kupffer cells. However, as with resting B cells, H3 hypomethylationwas reversed by mitotic stimulation; following overnight culture in thepresence of GM-SCF, CSF and interleukin 3 (IL-3), we observed highlevels of H3-K9 di/tri-methylation in CD45 positive Kupffer cells(compare upper panels, FIG. 5 c). These data provide strong evidencethat H3 methylation increases substantially as cells enter the cellcycle.

Discussion and Conclusions

Using a panel of antisera that recognise specific histone methylationstates we provide strong evidence of histone hypomethylation in G₀ Blymphocytes. Following mitotic stimulation, histone H3 methylation oflysines 4, 9 and 27 was reinstated in these cells, concurrent with anupregulation and redistribution of several chromatin modifier proteins.Although we cannot exclude the possibility that the lack of observedhistone methylation in these G₀ populations is due to an unusualchromatin conformation obstructing the recognition of methylationepitopes, we view this possibility as extremely unlikely for severalreasons. Firstly, antisera to different molecular epitopes showed aconsistent reduction in histone methylation as judged by IF. Secondly,this observation was confirmed by western blotting—an approach wheresteric masking of epitopes is not an issue. Thirdly, reduced anti-methylH3 labeling of resting cells was observed even within presumedeuchromatin (recognised by H3-K4) although these regions would begenerally considered to be relatively decondensed and accessible. Wetherefore favor the hypothesis that histone methylation is “lost” ordramatically reduced in quiescent B cells and also in Kupffer cells inmouse liver. An important question is how this loss might be achieved.Several reports have shown that histones are continuously being replacedin cells and that replacement occurs in a replication dependent andindependent manner [41, 42]. Cells which remain quiescent for prolongedperiods without going through S-phase (for example neurons) have beenshown to selectively replace histone H3 with the variant histone H3.3that, unlike H3, is synthesised throughout the cell cycle [43, 44]. Apredominance of the H3.3 variant has been documented in mouse liver [45]and lymphocytes. In lymphocytes, detailed analysis of histonecomposition during lymphocyte activation suggests that the proportion ofH3.3 within the mass pattern of chromatin is directly linked to thelength of time in quiescence [46]. More recently, the exchange of H3.1for H3.3 has been shown at specific loci where replacement appears to befavored or driven by active transcription [47]. Our data would fit witha gradual exchange of H3.1 for H3.3 in quiescent lymphocytes, whichtogether with the lack of expression (or inactivity) of several SETdomain proteins (such as Ezh2 and ESET), could result in a globalreduction in histone methylation in long-term quiescent lymphocytepopulations.

One important consideration is whether the apparent loss of histonemethylation that occurs as activated lymphocytes exit the cell cycle, isfunctionally significant. For example, a consequence of reduced histonemethylation in resting cells might be to ‘loosen’ the epigenetic codeand effectively enhance cellular plasticity. In principle this couldoffer an explanation for longstanding claims that some resting (orserum-starved) populations of cells are more efficiently reprogrammedthan activated cells [48-50]. To test whether resting B cells arereprogrammed at a higher frequency than activated B cells, we performednuclear transfer experiments using fertilized embryos as recipients.This allows the potential of different donor nuclei to be assessed in acontext where their contribution to embryonic development is notrequired [51]. As an indicator of plasticity, we compared the extent towhich an EGFP transgene [52, 53] that is silent in both resting andactive B cells but active from the morula stage onwards (our unpublisheddata), becomes reactivated after the transfer of lymphocyte nuclei intoone-cell embryos.

Nuclei from resting (0-24 hours) or active (48-72 hours) B cellscarrying the silent EGFP transgene were transferred into fertilisedembryos 18-21 hours post injection with human chorionic gonadotrophin(BL6/D2×BL6/D2). Embryos surviving the transfer procedure were culturedovernight in M16 media (Specialty Media). On day one after transfer,2-cell embryos were transferred into glucose-supplemented CZB media. Themajority of operated embryos reached morulae or early blastocyst stagebut were somewhat developmentally retarded as compared to non-operated,control embryos. On day four after transfer, GFP expression wasassessed. Re-expression of GFP occurred in twice as many fertilizedembryos injected with resting B cell nuclei as those injected with 48-or 72-hour activated B cells (FIG. 9). This enhanced performance did notsimply reflect a decline in viability of cultured cells since G₀ cellsmaintained for 24 hours in IL-4 alone (in the absence of mitoticstimulation and upregulation of histone methylation), also showedefficient reversal of transgene silencing (compare 12-16% versus 6-9%GFP-positive embryos, FIG. 9). The data from various experiments isshown in the table in FIG. 9. Similar preliminary results have beenobtained with resting and active T cells. These experiments suggest thathistone hypomethylation could contribute to the enhanced genomicplasticity of resting cells.

The observation that histone acetylation is robustly retained byquiescent cells whereas histone methylation appears to be a relativelyunstable epigenetic trait is intriguing. An explanation for this couldbe that the basal transcription of active genes in G₀ lymphocytes issufficient to maintain acetylation of the genome. High levels of histonemethylation, in contrast, may only be required only when overall geneactivity is increased (for example following mitotic stimulation) toamplify the epigenetic status of a gene prior to DNA synthesis. Thefinding that histone methylation is a more dynamic epigenetic imprintthan was previously anticipated is important. Current views of how ahistone code might be interpreted have implied that quality and densityof different histone tail modifications in a particular region could bepredictive of transcriptional potential. This assumption forms the basisfor current Chromatin Immunoprecipitation (ChIP)-based analyses. Here weshow that the relative abundance of histone H3 methylation in primarycells differs dramatically between resting and cycling populations. Thisfact does not negate the concept that histone methylation contributes tocellular memory since relatively low levels of these modifications couldstill be sufficient to ‘mark’ active or inactive chromatin domains inquiescent cells. However, our demonstration that histone methylation ismodulated according to cell cycle status indicates that the density of aparticular histone modification cannot simply be equated withtranscriptional competence.

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1. A method of producing an animal embryo, the method comprisingtransferring from a nuclear donor cell which has been selected on thebasis that it is histone hypomethylated at least a portion of thenuclear contents including at least the minimum chromosomal materialable to support development into a suitable recipient cell.
 2. Themethod of claim 1 wherein the nuclear donor cell has been selected byexperimentally determining that a first cell is histone hypomethylatedand selecting a second cell to be which is similar or identical to thefirst cell to thereby select a histone hypomethylated cell to be used assaid nuclear donor cell.
 3. The method of claim 2 wherein said firstcell and second cell are from the same population of cells.
 4. Themethod of claim 1 wherein the nuclear donor cell has been selected byselecting a cell of a type which has been previously determined as beinghistone hypomethylated or which has been previously determined as beinglikely to be histone hypomethylated.
 5. The method of claim 2 whereinthe level of histone methylation of said first cell or of said cell typewhen histone hypomethylated is negligible or absent.
 6. The method ofclaim 2 wherein the level of histone methylation of said first cell orof said cell type when histone hypomethylated is assessed on the basisof methylation at one or more residues of H3.
 7. The method of claim 2wherein the level of histone methylation of said first cell or of saidcell type when histone hypomethylated is assessed on the basis ofmethylation at one or more lysine residues.
 8. The method according toclaim 7 wherein the level of histone methylation is assessed on thebasis of methylation at one, two, three or four of the following lysineresidues: residues H3, H3, H3K2′ and H3.
 9. The method according toclaim 8 wherein the level of histone methylation is assessed on thebasis of methylation atH3K4 and H3K9.
 10. The method according to claim1 wherein the nuclear donor cell is a mammalian cell.
 11. The methodaccording to claim 1 wherein the recipient cell is a mammalian cell. 12.The method according to claim 1 wherein the recipient cell is anenucleated oocyte.
 13. A method of producing an animal embryo, themethod comprising transferring from a nuclear donor cell at least aportion of the nuclear contents including at least the minimumchromosomal material able to support development into a suitablerecipient cell wherein the nuclear donor cell is obtained from an embryoobtained by the method of claim
 1. 14. The method according to claim 13wherein the nuclear donor cell has been selected on the basis that it ishistone hypomethylated.
 15. A method of producing a foetus, the methodcomprising allowing an embryo obtained by a method according to claim 1to develop into a foetus.
 16. A method of producing a non-human animalthe method comprising allowing an embryo obtained by a method accordingto claim 1 to develop into said non-human animal.
 17. A method ofproducing an embryonic stem cell line, the method comprisingtransferring an embryo obtained by the method of claim 1 to a culturesystem.
 18. A method of producing an embryonic stem cell line, themethod comprising isolating the inner cell mass of an embryo obtained bythe method of claim 1 and transferring the inner cell mass to a culturesystem.
 19. A method according to claim 1 wherein the nuclear donor cellis a non-human cell.
 20. A method according to claim 1 wherein therecipient cell is a non-human cell. 21-33. (canceled)